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United States Patent | 6,010,893 |
Kihira | January 4, 2000 |
The present invention relates to a process for producing a human matrilysin characterized in that a human promatrilysin is expressed in E. coli and secreted into the periplasm thereof; the inclusion body is formed, the human promatrilysin is solubilized with a urea solution, purified and renatured to obtain the active enzyme (active-type matrilysin). By this process, the active human matrilysin can be easily produced from transformed E. coli.
Inventors: | Kihira; Yasunori (Shiga-ken, JP) |
Assignee: | Oriental Yeast Co., Ltd. (Tokyo, JP) |
Appl. No.: | 896062 |
Filed: | July 17, 1997 |
Sep 30, 1994[JP] | 6-259576 |
Current U.S. Class: | 435/226; 435/212; 435/219; 435/252.3; 435/252.33; 435/320.1; 536/23.1; 536/23.2; 536/23.5 |
Intern'l Class: | C12N 009/64; C07H 021/04 |
Field of Search: | 435/226,219,212,252.3,252.33,320.1 536/23.1,23.2,23.4,23.5 |
4652639 | Mar., 1987 | Stabinsky | 536/27. |
5240831 | Aug., 1993 | Barnes | 435/69. |
Sambrook et al. Molecular Cloning: A laboratory Manual, second edition, Cold Spring Harbor Laboratory Press, p. 17.11, 1989. Grosjean et al. "Preferential codon usage in prokaryotic genes: the optimal codon-anticodon interaction energy and the selective . . . " Gene 18, 199-209, 1982. Solar et a. "Zinc content of promatrilysin . . . " Biochem. Biophys. Res. comm. 201, 917-923, Jun. 15, 1994. Barnett et al. "Prod., purif., and charact. of human Matrilysin . . . " Protein Exp. and Pur. 5, 27-46, Feb. 1994. Crabbe et al. "Biochem. charct. of Matrilysin. Activation . . . " Biochemistry 31, 8500-8507, 1992. Muller et al. "The collagenase gene family in human consists of at . . . " Biochem. J. 253, 187-192, 1988. Marti et al. "Molecular charact. of a low-molecular-mass matrix . . . " Biochem. J. 285, 899-905, 1992. Fujimura et al. "Secretion of recombinant ribonuclease . . . " FEBS 265, 71-74, 1990. Ye et al, Gene Synthesis and Expression in E. coli for Pump a Human Matrix Metalloproteinase, Biochemical and Biophysical Research Communications, vol. 186 No. 1 pp. 143-149, Jul. 5, 1992. Quantin et al, Pump-1 cDNA Codes for a Protein with Characteristics Similar to those of Classical Collagenase Family Members, Biochemistry, vol. 28, pp. 5327-5334, 1989. Miyazaki et al, Purification and Characterization of Extracellular Matrix-degrading Metalloproteinase, Matrin (Pump-1), Secreted from Human Rectal Carcinoma Cell Line Cancer Research, vol. 50, pp. 7758-7764, Dec. 15, 1990. Woessner et al, Purification and Properties of a Small Latent Matrix Metalloproteinase of the Rat Uterus, The Journal of Biological Chemistry, vol. 263 No. 34, pp. 16918-16925 Nov. 15, 1988. Muller et al, The Collagenase Gene Family in Humans Consists of at least Four Members Biochem. Journal, vol. 253 pp. 187-192, 1988. Daniele Muller et al, "The Collagenase Gene Family in Humans Consists of at Least Four Members." The Biochemistry Journal, vol. 253, No. 1, Jul. 1988, pp. 187-192. Dulce Soler et al, "Zinc Contect of Promatrilysin, Matrilysin and the Stromelysin Catalytic Domain." Biochemical and Biophysical Research Communications, vol. 201, No. 2, Jun. 15, 1992, pp. 917-923. Hiroyuki Yamamoto et al, "Expression of Matrilysin mRNA in Colorectal Adenomas and its Induction By Truncated Fibronectin." vol. 201, No. 2, Jun. 15, 1994, pp. 657-664. Donald Busiek et al, "The Matrix Metalloprotease Matrilysin (Pump) is Expressed in Developing Human Mononuclear Phagocytes." The Journal of Biological Chemistry, vol. 267, No. 13, May 5, 1992, pp. 9087-9092. Hans-Peter Marti et al, "Molecular Characterization of a Low-Molecular-Mass Matrix Metalloproteinase Secreted by Glomerular Mesangial Cells as Pump-1." The Biochemical Journal, vol. 285, No. 3, Aug. 1, 1992, pp. 899-905. Mireille Gaire et al, "Structure and Expression of the Human Gene for the Matrix Metalloproteinase Matrilysin." Journal of Biological Chemistry, vol. 269, No. 3, Jan. 21, 1994, pp. 2032-2040. |
TABLE 1 ______________________________________ SEQUENCE LISTING: ______________________________________ SEQ ID NO:1 Length of sequence: 825 Type of sequence: nucleic acid Type of strand: double strand Topology: linear Type of sequence: synthetic DNA Origin: Name of organism: human being Characteristics of sequence: Symbol indicating characteristics: CDS Location: 6 . . . 818 Method of determining characteristics: S Symbol indicating characteristics: sig peptide Location: 6 . . . 68 Method of determining characteristics: S Sequence ______________________________________
TABLE 2 __________________________________________________________________________ AATTCATGAAACAAAGCACTATTGCACTGGCACTCTTACCGTTACTGTTTACCCCTGTGACC 62 MetLysGlnSerThrIleAlaLeuAlaLeuLeuProLeuLeuPheThrProValThr 50 AAGGCCCTGCCGCTGCCGCAAGAAGCTGGTGGCATGTCTGAACTGCAGTGGGAACAGGCACAG 125 LysAlaLeuProLeuProGlnGluAlaGlyGlyMetSerGluLeuGlnTrpGluGlnAlaGln 1 5 10 15 GACTATCTGAAGCGTTTTTACCTGTACGACTCTGAAACCAAAAACGCTAATTCTCTTGAAGCT 188 AspTyrLeuLysArgPheTyrLeuTyrAspSerGluThrLysAsnAlaAsnSerLeuGluAla 20 25 30 35 40 AAACTGAAGGAGATGCAGAAATTTTTCGGTCTGCCGATCACCGGTATGCTGAACTCCCGTGTT 251 LysLeuLysGluMetGlnLysPhePheGlyLeuProIleThrGlyMetLeuAsnSerArgVal 45 50 55 60 ATCGAAATCATGCAGAAACCGCGTTGTGGTGTTCCGGACGTTGCTGAATACTCTCTGTTCCCG 314 IleGluIleMetGlnLysProArgCysGlyValProAspValAlaGluTyrSerLeuPhePro 65 70 75 80 AACTCTCCGAAATGGACCTCTAAAGTTGTAACCTACCGTATCGTTTCTTACACCCGTGACCTG 377 AsnSerProLysTrpThrSerLysValValThrTyrArgIleValSerTyrThrArgAspLeu 85 90 95 100 CCGCATATCACCGTTGACCGTCTGGTTTCTAAAGCTTTGAACATGTGGGGTAAAGAGATCCCG 440 ProHisIleThrValAspArgLeuValSerLysAlaLeuAsnMetTrpGlyLysGluIlePro 105 110 115 120 CTGCATTTTCGTAAAGTTGTATGGGGTACCGCTGACATTATGATCGGTTTCGCTCGTGGTGCT 503 LeuHisPheArgLysValValTrpGlyThrAlaAspIleMetIleGlyPheAlaArgGlyAla 125 130 135 140 145 CATGGTGACTCTTACCCGTTCGACGGCCCGGGTAACACCCTGGCGCATGCTTTCGCTCCGGGT 566 HisGlyAspSerTyrProPheAspGlyProGlyAsnThrLeuAlaHisAlaPheAlaProGly 150 155 160 165 ACTGGTCTGGGTGGCGACGCACACTTCGACGAAGACGAACGTTGGACCGACGGTTCTTCCCTG 629 ThrGlyLeuGlyGlyAspAlaHisPheAspGluAspGluArgTrpThrAspGlySerSerLeu 170 175 180 185 GGTATCAACTTCCTGTACGCTGCAACTCATGAACTGGGTCATTCTCTGGGCATGGGTCATTCT 692 GlyIleAsnPheLeuTyrAlaAlaThrHisGluLeuGlyHisSerLeuGlyMetGlyHisSer 190 195 200 205 TCCGACCCGAACGCTGTTATGTACCCGACCTACGCTAACGGTGACCCGCAGAACTTCAAACTG 755 SerAspProAsnAlaValMetTyrProThrTyrGlyAsnGlyAspProGlnAsnPheLysLeu 210 215 220 225 TCTCAGGACGATATCAAAGGTATCCAGAAACTGTACGGTAAACGTTCTAACTCTCGTAAAAAG 818 SerGlnAspAspIleLysGlyIleGlnLysLeuTyrGlyLysArgSerAsnSerArgLysLys 230 235 240 245 250 TAATAGG 825 __________________________________________________________________________
__________________________________________________________________________ # SEQUENCE LISTING - (1) GENERAL INFORMATION: - (iii) NUMBER OF SEQUENCES: 2 - (2) INFORMATION FOR SEQ ID NO:1: - (i) SEQUENCE CHARACTERISTICS: #pairs (A) LENGTH: 825 base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear - (ii) MOLECULE TYPE: cDNA - (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 6..818 #ID NO:1: (xi) SEQUENCE DESCRIPTION: SEQ #CTC TTA CCG TTA CTG 47 GCA CTG GCA #Leu Ala Leu Leu Pro Leu Leue Ala # 10 - TTT ACC CCT GTG ACC AAG GCC CTG CCG CTG CC - #G CAA GAA GCT GGT GGC 95 Phe Thr Pro Val Thr Lys Ala Leu Pro Leu Pr - #o Gln Glu Ala Gly Gly # 30 - ATG TCT GAA CTG CAG TGG GAA CAG GCA CAG GA - #C TAT CTG AAG CGT TTT 143 Met Ser Glu Leu Gln Trp Glu Gln Ala Gln As - #p Tyr Leu Lys Arg Phe # 45 - TAC CTG TAC GAC TCT GAA ACC AAA AAC GCT AA - #T TCT CTT GAA GCT AAA 191 Tyr Leu Tyr Asp Ser Glu Thr Lys Asn Ala As - #n Ser Leu Glu Ala Lys # 60 - CTG AAG GAG ATG CAG AAA TTT TTC GGT CTG CC - #G ATC ACC GGT ATG CTG 239 Leu Lys Glu Met Gln Lys Phe Phe Gly Leu Pr - #o Ile Thr Gly Met Leu # 75 - AAC TCC CGT GTT ATC GAA ATC ATG CAG AAA CC - #G CGT TGT GGT GTT CCG 287 Asn Ser Arg Val Ile Glu Ile Met Gln Lys Pr - #o Arg Cys Gly Val Pro # 90 - GAC GTT GCT GAA TAC TCT CTG TTC CCG AAC TC - #T CCG AAA TGG ACC TCT 335 Asp Val Ala Glu Tyr Ser Leu Phe Pro Asn Se - #r Pro Lys Trp Thr Ser #110 - AAA GTT GTA ACC TAC CGT ATC GTT TCT TAC AC - #C CGT GAC CTG CCG CAT 383 Lys Val Val Thr Tyr Arg Ile Val Ser Tyr Th - #r Arg Asp Leu Pro His # 125 - ATC ACC GTT GAC CGT CTG GTT TCT AAA GCT TT - #G AAC ATG TGG GGT AAA 431 Ile Thr Val Asp Arg Leu Val Ser Lys Ala Le - #u Asn Met Trp Gly Lys # 140 - GAG ATC CCG CTG CAT TTT CGT AAA GTT GTA TG - #G GGT ACC GCT GAC ATT 479 Glu Ile Pro Leu His Phe Arg Lys Val Val Tr - #p Gly Thr Ala Asp Ile # 155 - ATG ATC GGT TTC GCT CGT GGT GCT CAT GGT GA - #C TCT TAC CCG TTC GAC 527 Met Ile Gly Phe Ala Arg Gly Ala His Gly As - #p Ser Tyr Pro Phe Asp # 170 - GGC CCG GGT AAC ACC CTG GCG CAT GCT TTC GC - #T CCG GGT ACT GGT CTG 575 Gly Pro Gly Asn Thr Leu Ala His Ala Phe Al - #a Pro Gly Thr Gly Leu 175 1 - #80 1 - #85 1 - #90 - GGT GGC GAC GCA CAC TTC GAC GAA GAC GAA CG - #T TGG ACC GAC GGT TCT 623 Gly Gly Asp Ala His Phe Asp Glu Asp Glu Ar - #g Trp Thr Asp Gly Ser # 205 - TCC CTG GGT ATC AAC TTC CTG TAC GCT GCA AC - #T CAT GAA CTG GGT CAT 671 Ser Leu Gly Ile Asn Phe Leu Tyr Ala Ala Th - #r His Glu Leu Gly His # 220 - TCT CTG GGC ATG GGT CAT TCT TCC GAC CCG AA - #C GCT GTT ATG TAC CCG 719 Ser Leu Gly Met Gly His Ser Ser Asp Pro As - #n Ala Val Met Tyr Pro # 235 - ACC TAC GGT AAC GGT GAC CCG CAG AAC TTC AA - #A CTG TCT CAG GAC GAT 767 Thr Tyr Gly Asn Gly Asp Pro Gln Asn Phe Ly - #s Leu Ser Gln Asp Asp # 250 - ATC AAA GGT ATC CAG AAA CTG TAC GGT AAA CG - #T TCT AAC TCT CGT AAA 815 Ile Lys Gly Ile Gln Lys Leu Tyr Gly Lys Ar - #g Ser Asn Ser Arg Lys 255 2 - #60 2 - #65 2 - #70 # 825 Lys - (2) INFORMATION FOR SEQ ID NO:2: - (i) SEQUENCE CHARACTERISTICS: #acids (A) LENGTH: 271 amino (B) TYPE: amino acid (D) TOPOLOGY: linear - (ii) MOLECULE TYPE: protein - (xi) SEQUENCE DESCRIPTION: - # SEQ ID NO:2: - Met Lys Gln Ser Thr Ile Ala Leu Ala Leu Le - #u Pro Leu Leu Phe Thr # 15 - Pro Val Thr Lys Ala Leu Pro Leu Pro Gln Gl - #u Ala Gly Gly Met Ser # 30 - Glu Leu Gln Trp Glu Gln Ala Gln Asp Tyr Le - #u Lys Arg Phe Tyr Leu # 45 - Tyr Asp Ser Glu Thr Lys Asn Ala Asn Ser Le - #u Glu Ala Lys Leu Lys # 60 - Glu Met Gln Lys Phe Phe Gly Leu Pro Ile Th - #r Gly Met Leu Asn Ser # 80 - Arg Val Ile Glu Ile Met Gln Lys Pro Arg Cy - #s Gly Val Pro Asp Val # 95 - Ala Glu Tyr Ser Leu Phe Pro Asn Ser Pro Ly - #s Trp Thr Ser Lys Val # 110 - Val Thr Tyr Arg Ile Val Ser Tyr Thr Arg As - #p Leu Pro His Ile Thr # 125 - Val Asp Arg Leu Val Ser Lys Ala Leu Asn Me - #t Trp Gly Lys Glu Ile # 140 - Pro Leu His Phe Arg Lys Val Val Trp Gly Th - #r Ala Asp Ile Met Ile 145 1 - #50 1 - #55 1 - #60 - Gly Phe Ala Arg Gly Ala His Gly Asp Ser Ty - #r Pro Phe Asp Gly Pro # 175 - Gly Asn Thr Leu Ala His Ala Phe Ala Pro Gl - #y Thr Gly Leu Gly Gly # 190 - Asp Ala His Phe Asp Glu Asp Glu Arg Trp Th - #r Asp Gly Ser Ser Leu # 205 - Gly Ile Asn Phe Leu Tyr Ala Ala Thr His Gl - #u Leu Gly His Ser Leu # 220 - Gly Met Gly His Ser Ser Asp Pro Asn Ala Va - #l Met Tyr Pro Thr Tyr 225 2 - #30 2 - #35 2 - #40 - Gly Asn Gly Asp Pro Gln Asn Phe Lys Leu Se - #r Gln Asp Asp Ile Lys # 255 - Gly Ile Gln Lys Leu Tyr Gly Lys Arg Ser As - #n Ser Arg Lys Lys # 270 __________________________________________________________________________